Expression and Solution NMR Study of Multi-site Phosphomimetic Mutant BCL-2 Protein

Author(s): Ting Song, Keke Cao, Yu dan Fan, Zhichao Zhang*, Zong W. Guo, Min H. Zhang, Peng Liu

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 6 , 2019

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background: The significance of multi-site phosphorylation of BCL-2 protein in the flexible loop domain remains controversial, in part due to the lack of structural biology studies of phosphorylated BCL-2.

Objective: The purpose of the study is to explore the phosphorylation induced structural changes of BCL-2 protein.

Methods: We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEEBCL- 2-EK (62-206)), in which the BH4 domain and the part of loop region was truncated (residues 2-61) to enable a backbone resonance assignment. The phosphorylation-induced structural change was visualized by overlapping a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy between EEE-BCL-2-EK (62-206) and BCL-2.

Results: The EEE-BCL-2-EK (62-206) protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from non-phosphorylated BCL-2 (npBCL-2) protein. Some residues in BH3 binding groove occurred chemical shift in the EEEBCL- 2-EK (62-206) spectrum, indicating that the phosphorylation in the loop region induces a structural change of active site.

Conclusion: The phosphorylation of BCL-2 induced structural change in BH3 binding groove.

Keywords: Phosphorylated BCL-2, purification, NMR spectroscopy, structural change, chemical shift, BH3 binding groove.

Song, T.; Chai, G.; Liu, Y.; Yu, X.; Wang, Z.; Zhang, Z. BCL-2 phosphorylation confers resistance on chronic lymphocytic leukaemia cells to the BH3 mimetics ABT-737, ABT-263 and ABT-199 by impeding direct binding. Br. J. Pharmacol., 2016, 173, 471-483.
Konopleva, M.; Contractor, R.; Tsao, T.; Samudio, I.; Ruvolo, P.P.; Kitada, S.; Verhaegen, M. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell, 2006, 10, 375-388.
Song, T.; Yu, X.; Liu, Y.; Li, X.; Chai, G.; Zhang, Z. (2015) Discovery of a small-molecule pBCL-2 inhibitor that overcomes pBCL-2-mediated resistance to apoptosis. ChemBioChem, 2015, 16, 757-765.
May, W.S.; Tyler, P.G.; Ito, T.; Armstrong, D.K.; Qatsha, K.A.; Davidson, N.E. Interleukin-3 and bryostatin-1 mediate hyperphosphorylation of BCL2 alpha in association with suppression of apoptosis. J. Biol. Chem., 1994, 269, 26865-26870.
Ito, T.; Deng, X.; Carr, B.; May, W.S. BCL-2 phosphorylation required for anti-apoptosis function. J. Biol. Chem., 1997, 272, 11671-11673.
Shitashige, M.; Toi, M.; Yano, T.; Shibata, M.; Matsuo, Y.; Shihasaki, F. Dissociation of Bax from a Bc1-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bc1-2. J. Biochem., 2001, 130, 741-748.
Deng, X.; Gao, F.; Flagg, T.; May, W.S. Mono-and multisite phosphorylation enhances BCL2's antiapoptotic function and inhibition of cell cycle entry functions. Proc. Natl. Acad. Sci. USA, 2004, 101, 153-158.
Petros, A.M.; Medek, A.; Nettesheim, D.G.; Kim, D.H.; Yoon, H.S.; Swift, K.; Fesik, S.W. Solution structure of the antiapoptotic protein BCL-2. Proc. Natl. Acad. Sci. USA, 2001, 98, 3012-3017.
Maiuri, M.C.; Criollo, A.; Tasdemir, E.; Vicencio, J.M.; Tajeddine, N.; Hickman, J.A.; Kroemer, G. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and BCL-2/BCL-XL. Autophagy, 2007, 3, 374-376.
Wei, Y.; Pattingre, S.; Sinha, S.; Bassik, M.; Levine, B. JNK1-mediated phosphorylation of BCL-2 regulates starvation-induced autophagy. Mol. Cell, 2008, 30, 678-688.
Zhang, Z.; Liu, Y.; Song, T.; Xue, Z.; Shen, X.; Liang, F.; Sheng, H. An antiapoptotic BCL-2 family protein index predicts the response of leukaemic cells to the pan-BCL-2 inhibitor S1. Br. J. Cancer, 2013, 108, 1870-1878.
Whitmore, L.; Wallace, B.A. Dichroweb, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res., 2004, 32(Suppl. 2), W668-W673.
Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Wang, S. Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal. Chem., 2004, 332, 261-273.
Vance, B.A.; Zacharchuk, C.M.; Segal, D.M. Recombinant Mouse BCL-2 (1-203) two domain connected by a long protease-sensitive linker. J. Biol. Chem., 1998, 271, 30811-30815.
Sato, T.; Hanada, M.; Bodrug, S.; Irie, S.; Iwama, N.; Boise, L.H.; Wang, H.G. Interactions among members of the BCL-2 protein family analyzed with a yeast two-hybrid system. Proc. Natl. Acad. Sci. USA, 1994, 91, 9238-9242.
Yang, J.S.; Hour, M.J.; Huang, W.W.; Lin, K.L.; Kuo, S.C.; Chung, J.G. MJ-29 inhibits tubulin polymerization, induces mitotic arrest, and triggers apoptosis via cyclin-dependent kinase 1-mediated BCL-2 phosphorylation in human leukemia U937 cells. Ther. Pharmacol. Exp. J., 2010, 334, 477-488.
Dai, H.; Ding, H.; Meng, X.W.; Lee, S.H.; Schneider, P.A.; Kaufmann, S.H. Contribution of BCL-2 phosphorylation to Bak binding and drug resistance. Cancer Res., 2013, 73, 6998-7008.
Perez-Galan, P.; Roue, G.; Lopez-Guerra, M.; Nguyen, M.; Villamor, N.; Montserrat, E.; Colomer, D. BCL-2 phosphorylation modulates sensitivity to the BH3 mimetic GX15-070 (Obatoclax) and reduces its synergistic interaction with bortezomib in chronic lymphocytic leukemia cells. Leukemia, 2008, 22, 1712-1720.
Hanada, M.; Aimé-Sempé, C.; Sato, T.; Reed, J.C. Structure-function analysis of BCL-2 protein identification of conserved domains important for homodimerization with BCL-2 and heterodimerization with Bax. J. Biol. Chem., 1995, 270, 11962-11969.
Huang, D.C.S.; Adams, J.M.; Cory, S. The conserved N-terminal BH4 domain of BCL-2 homologues is essential for inhibition of apoptosis and interaction with CED-4. EMBO J., 1998, 17, 1029-1039.
21. Follis, A.V.; Llambi, F.; Kalkavan, H.; Yao, Y.; Phillips, A.H.; Park, C.G.; Kriwacki, R.W. Regulation of apoptosis by an intrinsically disordered region of Bcl-xL. Nat. Chem. Biol., 2018, 14, 458-465.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Published on: 03 July, 2019
Page: [449 - 457]
Pages: 9
DOI: 10.2174/0929866526666190327121225
Price: $65

Article Metrics

PDF: 25